CN108950068A - A kind of avian infectious bronchitis virus QX type strain identification detection kit - Google Patents
A kind of avian infectious bronchitis virus QX type strain identification detection kit Download PDFInfo
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Abstract
The present invention relates to technical field of biological, it is desirable to provide a kind of avian infectious bronchitis virus QX type strain identification detection kit.The kit includes two pairs of primers, namely for expanding the universal primer of infectious bronchitis virus and for the primer of specific amplification QX genotype strain；Wherein, for the sequence of the primer of specific amplification QX genotype strain such as shown in SEQ ID NO:5 and SEQ ID NO:6.The present invention establishes effective RT-PCR detection method by designing the specific primer of QX genotype, it is only necessary to can achieve the purpose that carry out parting detection to the IBV of QX genotype by a RT-PCR reaction.2 pairs of primers are added in the same PCR system under same PCR reaction condition, the IBV in clinical sample or chick embryo allantoic liquid can be effectively detected out, also can specifically distinguish the IBV of QX type genotype by the kit.The detection method rapidly and efficiently, accurately, all has a very big significance the selection and the quick prevention and control of IB of instructing vaccine.
Description
Technical field
The invention belongs to technical field of biological, and in particular to avian infectious bronchitis virus QX strain identifies detection
Kit.
Background technique
Infectious bronchitis of chicken (Infectious Bronchitis, IB) is by avian infectious bronchitis virus
Acute, highly contagious disease caused by (Infectious Bronchitis virus, IBV).The disease can encroach on exhaling for chicken
Desorption system, urogenital system and digestive system etc..It is one of the Important Infectious Diseases for seriously endangering world's aviculture.
Avian infectious bronchitis virus belongs to the more virales coronaviridae coronavirus genus of Buddhist nun.Its genome be regardless of
The positive chain RNA virus of segment, overall length about 27.6kb.Viral genome is followed successively by 5 ' -1a-1b-S1-S2-3a- from 5 ' ends to 3 ' ends
3b-3c-M-5a-5b-N-poly(A)-3'.IBV contains 4 kinds of major structural proteins: spike protein (S), memebrane protein (M), nucleocapsid
Albumen (N), small envelope protein (E).The antigenic determinant of IBV is predominantly located in S protein, and S protein can not only mediate retroviral and easily
Sense cell merges, and determines the virulence of virus, and determine the antigenicity of virus.IBV uses identical elder generation in transcription
The unique transcription mechanism for leading sequence transcription different fragments occurs the genome of IBV easily along with RNA enzyme lacks check and correction mechanism
Point mutation, missing, insertion and recombination eventually lead to the variation of IBV strain, and new serotype and genotype continuously emerges.Largely
Studies have shown that the S1 gene of IBV is variation frequency and the highest gene of degree of variation, IBV parting and something lost based on S1 gene
Coming into analysis becomes a big research hotspot in recent years.
By the strain that various IBV can be divided into different genotype after the S1 gene progress phylogenetic analysis to IBV.Mesh
It is preceding China it is generally popular be QX genotype IBV strain.The IBV QX type strain past is only popular in China in 1990s,
It has been widely current after 2000s national including Europe and Russia etc. in all over the world.Conventional vaccine to the infection of QX strain not
Protection can be provided.Therefore the prevalence of QX strain causes very huge economic loss to poultry husbandry.Clinically it is badly in need of to QX genotype
IBV strain carry out specific rapid differential diagnosis, to instruct the selection of vaccine in time, reduce and lost caused by IBV infection.
Summary of the invention
The technical problem to be solved by the present invention is to overcome deficiency in the prior art, provide a kind of avian infectious bronchus
Scorching virus QX type strain identifies detection kit.
In order to solve the above technical problems, solution of the invention is:
A kind of kit identifying detection for avian infectious bronchitis virus QX type strain, including following two pairs are provided
Primer: for expanding the universal primer of infectious bronchitis virus and for the primer of specific amplification QX genotype strain；
Its sequence is as follows:
General-F:5'-CCAAAGTGCCTTCAGACC-3'(is as shown in SEQ ID NO:3)
General-R:5'-GCTAGACCAAGCCATACC-3'(is as shown in SEQ ID NO:4)
QX-F:5'-GTCATTTCTGAGTCAGTTTGTG-3'(is as shown in SEQ ID NO:5)
QX-R:5'-TGCGTTAATACTAAGGGCTC-3'(is as shown in SEQ ID NO:6).
In the present invention, the kit includes following compositions:
(1) RNA extracts reagent: Trizol reagent；
(2) Reverse Transcription: 5 × reaction buffer, the dNTP Mix of 10mM, the random primer of 0.2 μ g/ μ l, 20U/ μ l
Rnase inhibitor, the reverse transcriptase of 200U/ μ l, Nuclease-free water；
(3) PCR reaction reagent: 2 × Taq Master Mix, sterilize distilled water；
(4) serotype specific primer: the concentration of the universal primer and QX genotype strain specific primer is 10 μM；
(5) positive control sample: the reverse transcription product cDNA of QX genotype strain nucleic acid extraction object；
(6) negative control sample: the reverse transcription product cDNA of normal chick embryo allantoic liquid extract RNA.
In the present invention, the positive control sample is the allantoic fluid extract RNA of QX genotype strain IBV-HF infection chicken embryo
Reverse transcription product cDNA.
Kit provided by the invention, application method the following steps are included:
(1) total serum IgE is extracted from swab to be measured, tissue or infection chick embryo allantoic liquid sample:
(less than 500 μ after the swab, tissue or the infection chick embryo allantoic liquid sample that add 500 μ l to handle well in 1.5ml EP pipe
L's can supply 500 μ l with sterilizing PBS), 500 μ l Trizol reagents are added, mixes well, is stored at room temperature 5min；Add 200 μ l
Chloroform mixes well 15s, is stored at room temperature 5min；4 DEG C, 12000rpm is centrifuged 10min；It takes supernatant (400-600 μ l) plus isometric
Isopropanol, it is slight to mix, be stored at room temperature 5min；4 DEG C, 12000rpm is centrifuged 10min；It discards supernatant, adds 75% ethyl alcohol of 1ml,
4 DEG C, 12000rpm is centrifuged 7min；It discards supernatant, precipitates dry 10-15min, add 10 μ l Nuclease-free water to dissolve, obtain
Total serum IgE.(note: pipette tips and EP pipe RNA used when extracting should be free of RNA enzyme)
(2) total serum IgE extracted using previous step carries out reverse transcription reaction as template.The system of reaction are as follows: 11 μ l total serum IgEs
Template, 5 × reaction buffer of 4 μ l, the random primer of the 0.2 μ g/ μ l of the dNTP Mix, 1 μ l of the 10mM of 2 μ l, 1 μ l 20U/ μ
The Rnase inhibitor of l, the reverse transcriptase of 1 μ l 200U/ μ l, totally 20 μ l.In 42 DEG C of isothermal reaction 1h after mixing, then 70 DEG C of perseverances
Temperature terminates reaction 10min, obtains cDNA.
(3) using the reverse transcription product cDNA as template, PCR amplification, the PCR system are carried out using two pairs of serotype specific primers
Are as follows: 2 × Taq Master Mix 12.5 μ l, 10 μM of general upstream and downstream primer (general-F, general-R) each 0.8 μ l, 10 μM of QX
Each 1 μ l of 0.4 μ l, cDNA template of genotyping primer (QX-F, QX-R) adds sterilizing distilled water (ddH2O) to 25 μ l.
The reaction condition of the PCR reaction system are as follows: 95 DEG C of initial denaturation 5min, subsequently into 95 DEG C of denaturation 30s, 55 DEG C are moved back
Fiery 30s, 72 DEG C of extension 30s circulations, totally 35 recycle, 72 DEG C of extensions 10min, 16 DEG C of 5min；
(4) PCR reaction solution is taken to carry out 1% agarose gel electrophoresis, judging result after EB dyeing.
In the present invention, two bands for obtaining about 200bp and 340bp are expanded to QX genotype IBV strain RT-PCR, to non-QX
Strain amplification only obtains mono- band of about 200bp.The PCR product that QX strain IBV-HF amplification obtains is SEQ ID NO:1 and SEQ
Two sections of nucleotide sequences shown in ID NO:2.
Compared with prior art, the beneficial effects of the present invention are:
1, the present invention establishes effective RT-PCR detection method by the specific primer of design QX genotype.It only needs to lead to
Crossing a RT-PCR reaction can achieve the purpose that carry out parting detection to the IBV of QX genotype.
2,2 pairs of primers are added in the same PCR system under same PCR reaction condition for the kit, can be effectively
It detects the IBV in clinical sample or chick embryo allantoic liquid, also can specifically distinguish the IBV of QX type genotype.
3, the detection method rapidly and efficiently, accurately, all has instruct vaccine very much selection and the quick prevention and control of IB
Big meaning.
Detailed description of the invention
Fig. 1 is that kit RT-PCR expands IBV-HF strain (QX type), the result of M41 (non-QX type).
In figure, M.100bp marker；1. negative control；2.M41；3.IBV-HF
Fig. 2 is the result of kit RT-PCR specific amplification IBV.
In figure, M.100bp marker；1. negative control；2-6.NDV, AIV H5 hypotype, AIV H7 hypotype, AIV H9 are sub-
Type, IBDV；7.M41；8.IBV-HF.
Fig. 3 is the verification result that kit detects selected strain.
In figure, M.100bp marker；1. negative control；2-6.NDV, AIV H5 hypotype, AIV H7 hypotype, AIV H9 are sub-
Type, IBDV；7.IBV-GX-1023 (LDT-3 type)；8.4/91；9-14.M41,H120,H52,ZJ971,JH06011,JH06111
(Mass type)；15-18.IBV-SD, IBV-QX-H120, IBV-GX-0808, IBV-HF (QX type).
Specific embodiment
The kit forms utilized in the present invention include:
(1) RNA extracts reagent: Trizol reagent；
(2) Reverse Transcription: 5 × reaction buffer, the dNTP Mix of 10mM, the random primer of 0.2 μ g/ μ l, 20U/ μ l
Rnase inhibitor, the reverse transcriptase of 200U/ μ l, Nuclease-free water.
(3) PCR reaction reagent: 2 × Taq Master Mix, sterilize distilled water.
(4) serotype specific primer: the universal primer and QX genotype strain specific primer (as shown in SEQ ID NO:3-6)
Concentration be 10 μM；
(5) positive control sample: the reverse transcription product cDNA of QX genotype strain nucleic acid extraction object；
(6) negative control sample: the reverse transcription product cDNA of normal chick embryo allantoic liquid extract RNA.
The application method of mentioned reagent box, successively includes the following steps:
(1) total serum IgE is extracted from swab to be measured, tissue or infection chick embryo allantoic liquid sample:
(less than 500 μ after the swab, tissue or the infection chick embryo allantoic liquid sample that add 500 μ l to handle well in 1.5ml EP pipe
L's can use sterilizing PBS dilution), 500 μ l Trizol reagents are added, mixes well, is stored at room temperature 5min；Add 200 μ l chloroforms,
15s is mixed well, 5min is stored at room temperature；4 DEG C, 12000rpm is centrifuged 10min；Take supernatant (400-600 μ l) plus in equal volume different
Propyl alcohol, it is slight to mix, it is stored at room temperature 5min；4 DEG C, 12000rpm is centrifuged 10min；It discards supernatant, adds 75% ethyl alcohol of 1ml, 4 DEG C,
12000rpm is centrifuged 7min；It discards supernatant, precipitates dry 10-15min, add 10 μ lNuclease-free Water water to dissolve, obtain
Total serum IgE.(note: pipette tips RNA used when extracting should be free of RNA enzyme in EP pipe)
(2) total serum IgE extracted using previous step carries out reverse transcription reaction, system are as follows: 11 μ l total serum IgE templates, 4 μ as template
5 × reaction buffer of l, the random primer of the 0.2 μ g/ μ l of the dNTP Mix, 1 μ l of the 10mM of 2 μ l, the Rnase of 1 μ l20U/ μ l
Inhibitor, the reverse transcriptase of 1 μ l 200U/ μ l, totally 20 μ l.In 42 DEG C of isothermal reaction 1h after mixing, then 70 DEG C of constant temperature are terminated anti-
10min is answered, cDNA is obtained.
(3) using the reverse transcription product cDNA as template, PCR amplification, the PCR system are carried out using two pairs of serotype specific primers
Are as follows: 2 × Taq Master Mix 12.5 μ l, 10 μM of general upstream and downstream primer (general-F, general-R) each 0.8 μ l, 10 μM of QX
Each 1 μ l of 0.4 μ l, cDNA template of genotyping primer (QX-F, QX-R) adds sterilizing distilled water (ddH2O) to 25 μ l.
The reaction condition of the PCR reaction system are as follows: 95 DEG C of initial denaturation 5min, subsequently into 95 DEG C of denaturation 30s, 55 DEG C are moved back
Fiery 30s, the circulation of 72 DEG C of extension 30s carry out 35 circulations, 72 DEG C of extensions 10min, 16 DEG C of 5min altogether；
(4) it amplified production electrophoresis detection: takes above-mentioned reaction product to carry out agarose gel electrophoresis, takes 5 μ l amplified production points
Sample is in 1% Ago-Gel, 250V electrophoresis 25min, gel imaging system observation.QX genotype strain RT-PCR amplification is obtained
Two bands for obtaining about 200bp and 340bp, only obtain mono- band of about 200bp to non-QX genotype strain.
(5) amplified production is sequenced: the specific band for taking positive control strain IBV-HF amplification to obtain send sequencing company to carry out
Sequencing determines.The PCR product that amplification obtains is two sections of nucleotide sequences shown in SEQ ID NO:1-2, and size is respectively 193bp
And 339bp；
(6) method of the design obtains similar result to other QX genotype, the amplification of non-QX genotype strain: amplification QX
Genotype strain obtains about two band of 200bp and 340bp or so, when expanding non-QX genotype strain, only obtains about 200bp mono-
Band.
It is described further below with reference to embodiment and attached drawing.It should be understood that these embodiments are for illustration purposes only, and
It is not used in and limits the scope of the invention.
[embodiment 1] RNA extracting
The processing of 1 test sample
The processing of tissue sample: taking the tissue for the easily separated IBV such as chicken tracheae, kidney that dies of illness, after being shredded with scissors according to
1:3 volume ratio is added sterilizing PBS and is fully ground.The tissue suspension being homogenized is frozen repeatedly in -20 DEG C of (or lower temperature)-room temperatures
12,000rpm is centrifuged 10min after melting 3 times, takes 200 μ l of supernatant in new sterile centrifugation tube.
The processing of swab samples: fresh throat or cloacal swab are added 1ml sterilizing PBS and discard after oscillation stirs evenly
Swab (is as far as possible collected the liquid in swab complete).Leachate 12,000rpm are centrifuged 10min, take 200 μ l of supernatant in new
In sterile centrifugation tube.
Allantoic fluid processing: the chick embryo allantoic liquid for collecting virus infection carries out 12,000rpm centrifugation 10min, takes supernatant 200
μ l is in new sterile centrifugation tube.
2 viral RNAs extract
After the swab, tissue or the infection chick embryo allantoic liquid sample that add 500 μ l to handle well in 1.5ml EP pipe, 500 μ are added
L Trizol reagent, mixes well, is stored at room temperature 5min；Add 200 μ l chloroforms, mixes well 15s, be stored at room temperature 5min；4 DEG C,
12000rpm is centrifuged 10min；500 μ l of supernatant is taken to add isometric isopropanol, it is slight to mix, it is stored at room temperature 5min；4 DEG C,
12000rpm is centrifuged 10min；It discards supernatant, adds 75% ethyl alcohol of 1ml, 4 DEG C, 12000rpm is centrifuged 7min；Supernatant is abandoned, it will be in pipe
Remaining liq wink from rear careful exhaustion (noticing that pipette tips not encounter precipitating), EP tube opening is put on clean paper and is dried
15min adds 10 μ l Nuclease-free water to dissolve, obtains total serum IgE.(note: pipette tips RNA used when extracting should be free of with EP pipe
RNA enzyme)
[embodiment 2] reverse transcription reaction (RT reaction)
(4) total serum IgE extracted using previous step carries out reverse transcription reaction, the system of reverse transcription reaction as template are as follows:
11 μ l total serum IgE templates, 5 × reaction buffer of 4 μ l, the 0.2 μ g/ μ l's of the dNTP Mix, 1 μ l of the 10mM of 2 μ l
Random primer, the Rnase inhibitor of 1 μ l 20U/ μ l, the reverse transcriptase of 1 μ l 200U/ μ l, totally 20 μ l.In 42 DEG C of perseverances after mixing
Temperature reaction 1h, then 70 DEG C of constant temperature terminate reaction 10min, obtain cDNA.It is stored in -20 DEG C
[embodiment 3] PCR reaction
1. design of primers
It collects on GeneBank in recent years in the IBV QX type strain of China's prevalence and the S1 gene of some QX plants of classics
Sequence amounts to 212.The S1 gene order totally 49 of the non-QX type strain of various different genotypes is chosen from GeneBank simultaneously
Item.Sequence alignment is carried out to this 261 S1 genes using DNAMAN software, uses primer-design software Premier Primer 5
According to the conserved regions design 1 of QX type strain S1 gene to primer (QX-F, QX-R), and this to primer on the strain of non-QX type not
It is conservative.Universal primer (logical-F, lead to-R, table 1) refers to the Master's thesis of Ling Jifei.Primer is by the prosperous biotechnology in the Hangzhou Chinese catalpa Qing Ke
Co., Ltd's synthesis, PAGE purifying.Primer information is as shown in table 1.
1 kit serotype specific primer information of table
2. prepared by positive criteria product
IBV-HF (QX type) strain is inoculated with 9-11 age in days SPF chicken embryo, 36-72h harvest infection chick embryo allantois after egg infectious
Liquid.After handling chick embryo allantoic liquid well by preceding method, total serum IgE is extracted from allantoic fluid, reverse transcription is at cDNA.
Then cDNA concentration is measured, -20 DEG C save backup.
3.PCR reaction system optimization
Use the cDNA of above-mentioned preparation as template, respectively in PCR program primer concentration, annealing temperature, extend when
Between, the conditions such as PCR cycle number optimize.
The result optimized to reaction system and amplification program is as follows:
In 25 μ LPCR reaction systems, 2 × Taq Master Mix, 12.5 μ l, (general-F leads to general upstream and downstream primer
With each 0.8 μ l, QX genotyping primer (QX-F, QX-R) of-R) each 0.4 μ l, cDNA template 1 μ l, ddH2O 9.1μl。
The reaction condition of the PCR reaction system are as follows: 95 DEG C of initial denaturation 5min, subsequently into 95 DEG C of denaturation 30s, 55 DEG C are moved back
Fiery 30s, the circulation of 72 DEG C of extension 30s, totally 35 recycle, 72 DEG C of extensions 10min, 16 DEG C of 5min；
Using the system after optimization to positive criteria product PCR amplification result as shown in Figure 1, QX type strain (IBV-HF) expands
Two bands of about 200bp and 340bp out, non-QX type IBV strain (M41) only amplify mono- band of about 200bp (Fig. 1).Amplification knot
Fruit is consistent with desired design.
4.PCR product sequence verification
The PCR product that positive criteria product is expanded recycles, and send sequencing company sequencing analysis, verifies the sequence of amplification
Column are completely the same with expected theoretical sequence, also show through BLAST analysis, and the sequence of amplification is respective segments.Specific strain
Amplified fragments sequence is shown in SEQ ID NO1-2.Also the 340bp of other 4 plants of QX genotype strains or so band is sequenced
Verifying, as a result Blast is analysis shows that have high similitude with QX type strain sequence.
The specificity verification of [embodiment 4] kit
Choosing common avian viral includes avian influenza virus (AIV) H5, H7 and H9 subtype virus, newcastle disease virus
(NDV), the chick embryo allantoic liquid or chorioallantoic membrane of infectious bursal disease virus (IBDV) infection, extract RNA, reverse transcription at cDNA,
PCR detection is carried out with the system that IBV parting detecting reagent has optimized.Specificity verification result is as shown in Fig. 2, for positive right
According to IBV-HF strain cDNA amplification arrived specificity purpose band, non-QX type strain, which also expands, has obtained purpose band,
And any band (Fig. 2) cannot be expanded to common avian viral AIV (H5, H7, H9 hypotype), NDV, IBDV.
The verifying of [embodiment 5] different genotype strain sample detection
The part strain for taking animal virology key lab of the Ministry of Agriculture of Zhejiang University to save and animal section of Guangxi University
The part strain cDNA that poultry diease research department of skill institute give is as identifying object.The strain of verifying include 4/91 type strain,
LDT-3 type strain (IBV-GX-1023), Mass type strain (M41, H120, H52, ZJ971, JH06011, JH06111), QX type
Strain (IBV-SD, IBV-QX-H120, IBV-GX-0808, IBV-HF) and common avian viral AIV (H5, H7, H9 hypotype),
NDV,IBDV.The nucleic acid extraction object of these virus strain infection's chick embryo allantoic liquids is detected with kit of the invention respectively, as a result QX base
Because type IBV strain amplifies the DNA band of 200bp and 340bp or so, non-QX type IBV strain amplifies the item of 200bp or so
Band, non-IBV strain do not amplify band (Fig. 3).Demonstrate the specificity and validity of IBV RT-PCR parting detecting reagent.
Sequence table
<110>Zhejiang University
<120>a kind of avian infectious bronchitis virus QX type strain identifies detection kit
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 193
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ccaaagtgcc tttaggcctc caaatggatg gcatttgcaa gggggtgctt atgcagtagt 60
gaattctact aattatacta gtaatgccga ttctgcaagt gggtgcactg ttggtattat 120
taaggacgtc tataatcaaa gtgcggcttc catagctatg acagcacctc ctcagggtat 180
ggcttggtct aag 193
<210> 2
<211> 339
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gtcatttctg ggtcagtttg tgtataaggc aagtgatttt atgtatgggt cttatcatcc 60
taggtgttct tttagaccag aaaccattaa tagtggttta tggtttaatt ccttgtcagt 120
ttctcttact tatggacccc tacagggagg gtgtaagcaa tctgttttta gtggtaaggc 180
aacgtgttgt tatgcctact cttataatgg ccctagagca tgtaaaggtg tttattcagg 240
tgaattaagc aagacttttg aatgtggatt gctggtttat gttactaaga gtgatggctc 300
tcgtatacaa actagaacgg agcccttagt attaacgca 339
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ccaaagtgcc ttcagacc 18
<210> 4
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gctagaccaa gccatacc 18
<210> 5
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gtcatttctg agtcagtttg tg 22
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tgcgttaata ctaagggctc 20
Claims (5)
1. a kind of kit for identifying detection for avian infectious bronchitis virus QX type strain, which is characterized in that the reagent
Box includes two pairs of primers, namely for expanding the universal primer of infectious bronchitis virus and being used for specific amplification QX base
Because of the primer of type strain；Wherein, the sequence for the primer of specific amplification QX genotype strain is as follows:
QX-F:5'-GTCATTTCTGAGTCAGTTTGTG-3'
QX-R:5'-TGCGTTAATACTAAGGGCTC-3'.
2. kit according to claim 1, which is characterized in that described for expanding the logical of infectious bronchitis virus
It is as follows with the sequence of primer:
General-F:5'-CCAAAGTGCCTTCAGACC-3'
General-R:5'-GCTAGACCAAGCCATACC-3'.
3. kit according to claim 1, which is characterized in that the kit includes following compositions:
(1) RNA extracts reagent: Trizol reagent；
(2) Reverse Transcription: 5 × reaction buffer, the dNTP Mix of 10mM, the random primer of 0.2 μ g/ μ l, 20U/ μ l
Rnase inhibitor, the reverse transcriptase of 200U/ μ l, Nuclease-free water；
(3) PCR reaction reagent: 2 × Taq Master Mix, sterilize distilled water；
(4) serotype specific primer: the concentration of the universal primer and QX genotype strain specific primer is 10 μM；
(5) positive control sample: the reverse transcription product cDNA of QX genotype strain nucleic acid extraction object；
(6) negative control sample: the reverse transcription product cDNA of normal chick embryo allantoic liquid extract RNA.
4. kit according to claim 3, which is characterized in that the positive control sample is QX genotype strain IBV-
HF infects the reverse transcription product cDNA of the allantoic fluid extract RNA of chicken embryo.
5. the application method of kit described in claim 1, which comprises the following steps:
(1) total serum IgE is extracted from swab to be measured, tissue or infection chick embryo allantoic liquid sample:
It is supplied after the swab, tissue or the infection chick embryo allantoic liquid sample that add 500 μ l to handle well in 1.5ml EP pipe with the PBS that sterilizes
To 500 μ l；500 μ l Trizol reagents are added, mixes well, is stored at room temperature 5min；Add 200 μ l chloroforms, mixes well 15s, room
Temperature stands 5min；4 DEG C, 12000rpm is centrifuged 10min；Supernatant 400-600 μ l is taken to add isometric isopropanol, slight to mix, room
Temperature stands 5min；4 DEG C, 12000rpm is centrifuged 10min；It discards supernatant, adds 75% ethyl alcohol of 1ml, 4 DEG C, 12000rpm is centrifuged
7min；It discards supernatant, precipitates dry 10-15min, add 10 μ l Nuclease-free water to dissolve, obtain total serum IgE；
(2) total serum IgE extracted using previous step carries out reverse transcription reaction as template；The system of reaction are as follows: 11 μ l total serum IgE templates,
5 × reaction buffer of 4 μ l, the random primer of the 0.2 μ g/ μ l of the dNTP Mix, 1 μ l of the 10mM of 2 μ l, 1 μ l 20U/ μ l's
Rnase inhibitor, the reverse transcriptase of 1 μ l 200U/ μ l, totally 20 μ l；In 42 DEG C of isothermal reaction 1h after mixing, then 70 DEG C of constant temperature
Reaction 10min is terminated, cDNA is obtained；
(3) using the reverse transcription product cDNA as template, PCR amplification, the PCR system are as follows: 2 are carried out using two pairs of serotype specific primers
× Taq Master Mix 12.5 μ l, 10 μM of general each 0.8 μ l of upstream and downstream primer, 10 μM of QX genotyping primers QX-F and QX-R
Each 1 μ l of 0.4 μ l, cDNA template adds sterilizing distilled water to 25 μ l；
The reaction condition of the PCR reaction system are as follows: 95 DEG C of initial denaturation 5min, subsequently into 95 DEG C of denaturation 30s, 55 DEG C of annealing
30s, 72 DEG C of extension 30s circulations, totally 35 recycle, 72 DEG C of extensions 10min, 16 DEG C of 5min；
(4) PCR reaction solution is taken to carry out 1% agarose gel electrophoresis, judging result after EB dyeing.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747292A (en) * | 2019-11-28 | 2020-02-04 | 青岛易邦生物工程有限公司 | Method for identifying avian infectious bronchitis QX type virus wild virus and vaccine virus |
| CN110779970A (en) * | 2019-09-18 | 2020-02-11 | 南京农业大学 | Electrochemical detection method for chicken infectious bronchitis virus H120 strain |
| CN111471801A (en) * | 2020-02-21 | 2020-07-31 | 扬州大学 | Reverse transcription-real-time fluorescence quantitative PCR kit for detecting TC07-2 avian infectious bronchitis virus and application thereof |
| CN114134255A (en) * | 2021-12-29 | 2022-03-04 | 山东农业大学 | Dual PCR primer group and kit for detecting novel raccoon parvovirus and application of dual PCR primer group and kit |
| CN116024388A (en) * | 2023-01-05 | 2023-04-28 | 福建圣泽生物科技发展有限公司 | RT-PCR detection kit for infectious bronchitis virus GVI-1 genotype and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239252A (en) * | 2008-08-08 | 2011-11-09 | 惠氏有限责任公司 | Infectious bronchitis vaccines derived from ib-qx-like strains |
| CN102899423A (en) * | 2012-10-23 | 2013-01-30 | 广西壮族自治区兽医研究所 | GeXP (Gene Expression) rapid detection kit capable of simultaneously identifying six virus of chicken respiratory disease |
| CN105039585A (en) * | 2015-03-08 | 2015-11-11 | 江苏省家禽科学研究所 | Primer used in RT-PCR detection of chicken infectious bronchitis virus, kit comprising the primer and detection method thereof |
| CN105463136A (en) * | 2016-01-18 | 2016-04-06 | 浙江大学 | Kit for RT-PCR typing detection of avian infectious bronchitis virus |
-
2018
- 2018-07-18 CN CN201810788384.6A patent/CN108950068B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239252A (en) * | 2008-08-08 | 2011-11-09 | 惠氏有限责任公司 | Infectious bronchitis vaccines derived from ib-qx-like strains |
| CN102899423A (en) * | 2012-10-23 | 2013-01-30 | 广西壮族自治区兽医研究所 | GeXP (Gene Expression) rapid detection kit capable of simultaneously identifying six virus of chicken respiratory disease |
| CN105039585A (en) * | 2015-03-08 | 2015-11-11 | 江苏省家禽科学研究所 | Primer used in RT-PCR detection of chicken infectious bronchitis virus, kit comprising the primer and detection method thereof |
| CN105463136A (en) * | 2016-01-18 | 2016-04-06 | 浙江大学 | Kit for RT-PCR typing detection of avian infectious bronchitis virus |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110779970A (en) * | 2019-09-18 | 2020-02-11 | 南京农业大学 | Electrochemical detection method for chicken infectious bronchitis virus H120 strain |
| CN110779970B (en) * | 2019-09-18 | 2022-04-12 | 南京农业大学 | Electrochemical detection method for chicken infectious bronchitis virus H120 strain |
| CN110747292A (en) * | 2019-11-28 | 2020-02-04 | 青岛易邦生物工程有限公司 | Method for identifying avian infectious bronchitis QX type virus wild virus and vaccine virus |
| CN111471801A (en) * | 2020-02-21 | 2020-07-31 | 扬州大学 | Reverse transcription-real-time fluorescence quantitative PCR kit for detecting TC07-2 avian infectious bronchitis virus and application thereof |
| CN114134255A (en) * | 2021-12-29 | 2022-03-04 | 山东农业大学 | Dual PCR primer group and kit for detecting novel raccoon parvovirus and application of dual PCR primer group and kit |
| CN116024388A (en) * | 2023-01-05 | 2023-04-28 | 福建圣泽生物科技发展有限公司 | RT-PCR detection kit for infectious bronchitis virus GVI-1 genotype and application thereof |
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